The nature of thymus immigrants that contribute most effectively to T lymphopoiesis is unknown even though several well-characterized progenitors have been described. Some of these cells can give rise to lymphoid and non-lymphoid lineages, others are lymphoid-restricted and yet others are T lymphoid lineage-restricted. Cells with T lineage-restricted potential, circulating T cell progenitors (CTP), have recently been described in the blood with the aid of a pre-TCR1- controlled reporter gene. Here it is proposed to further phenotypically characterize CTP and their progeny and to analyze their efficacy in T cell generation and commitment to the T cell lineage in further detail. Furthermore, the Notch dependence of T lineage commitment will be addressed in mice with reversible inhibition of Notch-dependent transcription by analyzing the generation of cells with a CTP surface phenotype from precursors that are deficient in Notch- dependent transcription. Subsequently, after isolation of CTP and relieving the transcriptional block, the functional potential will be determined. Finally, gene expression of CTP will be compared with that of multipotent (LSK) and intrathymic (ETP) precursors in an attempt to place these cells in a differentiation pathway. Special emphasis will be given to gene regulation by the LRF gene that antagonizes Notch in early precursors of lymphoid cells. In a second aim, the efficacy of several well-characterized extrathymic precursors to generate T cells will be compared by depleting such precursors prior to injecting bone marrow or blood cells into Il-7ra- deficient recipients. In additional studies lineage fate tracing by IL-7R1-cre will be used to determine the contribution of IL-7R1-expressing precursors to intrathymic precursors such as ETP. Furthermore, the short-term potential of cells to home to the thymus will be compared 24 hours after injection of cells by explanting intrathymic donor-derived cells under conditions of limiting dilution into cultures containing OP9-DL1 feeder cells. Once information on different precursors with either multipotential (prototype LSK), lymphoid-restricted potential (prototype CLP) or T lineage-restricted potential (prototype CTP) has been established these subsets will be further subdivided by already established markers in order to pinpoint the most relevant precursors as precisely as possible. Overall this project aims at establishing mechanisms of extrathymic T lineage commitment as well as identifying the most potent extrathymic precursors for T lymphopoiesis. PUBLIC HEALTH RELEVANCE: This project aims at the identification and characterization of the most effective cells in bone marrow and blood that under different conditions permit the reconstitution of the T cell immune system that protects from infection and malignancy. Identification of the extrathymic T cell precursors will help in the faster regeneration of the immune system after treatment of malignancy by x- irradiation and/or cytotoxic drugs that result in T cell depletion.